The field of this invention is microbial genetics, especially as related to immunogenic compositions comprising attenuated bacterial pathogens or components thereof.
The Brucella species are important zoonotic pathogens affecting a wide variety of mammals. In agriculturally important domestic animals, these bacteria cause abortion and infertility, and they are of serious economic concern worldwide (5). In humans, Brucella species constitute potential bio-warfare agents. Brucella species that infect humans cause in undulating fever, which if untreated, can manifest as orchitis, osteoarthritis, spondylitis, endocarditis, and neurological disorders (11, 46). Currently there is no vaccine to protect against human brucellosis, especially that caused by B. melitensis. Treatment of brucellosis requires a prolonged combination of antibiotic therapy and is still problematic because of the potential for relapse.
Identifying Brucella virulence factors has been of great interest in understanding Brucella pathogenesis and immune evasion. After entry into macrophages virulent Brucella cells reside in an acidified vacuole, the Brucella containing vacuole (BCV). The BCV transiently interacts with early endosomes, followed by VirB-dependent sustained interaction with the endoplasmic reticulum (7). Thus, the BCV matures into a replicative niche in a VirB-dependent manner (7, 8). VirB proteins forming the type IV secretion system (T4SS) constitute important factors for Brucella virulence and intracellular replication (9, 14, 34). Lipopolysaccharide (LPS) is also an important virulence factor (27). Brucella LPS has minimal endotoxic effect, blocks complement activation, and protects against bactericidal cationic peptides (28). The O-chain is also important for the conventional entry of Brucella into macrophages through lipid rafts, a route which avoids fusion of the BCV with lysosomes (33, 37). Cyclic β-1, 2 glucan has been shown as an important virulence factor required for intracellular survival of Brucella (3). Although T4SS, cyclic β-1, 2 glucan, and LPS are clearly virulence factors of Brucella, the attenuated mutants lacking these virulence factors are either considered not safe or insufficient information is available to use them as vaccines for humans. This has necessitated identification of additional vaccine targets.
Several genetic loci that are required for Brucella replication in vitro have been identified (14, 24). In vitro conditions may not adequately reflect in vivo infection, and therefore, findings may have little or no in vivo relevance (45). In vivo screening methods have been used to identify Brucella genes required for survival and persistence (18, 26), however, these previous studies have relied on the conventional approach of determining tissue-specific cell counts (CFU) from multiple animals at different times, a process that is labor intensive and requires large numbers of animals. Because infection is a dynamic process and varies within individual mice, monitoring disease progression temporally within the same mouse provides a more comprehensive picture of pathogenic events. Further, such real-time analysis may reveal virulence determinants responsible for tissue specific replication of bacteria that would not be revealed using conventional CFU enumeration from liver and spleen.
Bioluminescent imaging of mice allows direct visualization of the infection process and is highly useful for bacterial pathogenesis studies (10), because the intensity of bioluminescence strongly correlates with the number of bacteria in the infected organs (16, 40). Bioluminescent imaging is useful in analyzing sub-acute and chronic infections that are often difficult to assess using conventional approaches because of uncertain bacterial locations (16, 40).
There is a long felt need in the art for safe and effective vaccines that protect humans and animals from infection by the pathogenic Brucella species, especially B. melitensis. 